Specialized cell surface receptors (termed here selecting) on endothelial cells and various circulating cells are involved in a number of intercellular interactions. For instance, an adhesion molecule on the surface of leukocytes, lymphocyte homing receptor (LHR), is known to be involved in the adhesive interactions of leukocytes with the endothelial lining of blood vessels. This adhesive interaction is a prerequisite for the movement of leukocytes from the blood to tissue sites where immune reactions and inflammatory reactions occur.
LHR is a lectin-like protein which performs its adhesive function recognizing carbohydrate-containing ligands on endothelial cells. Lectin-like receptors have also been found on endothelial cells and platelets. Endothelial leukocyte adhesion molecule-1 (ELAM-1) also known as CD62E and E-selectin is present on endothelial cells and is involved in the recognition of various circulating cells by the endothelium. Granule membrane protein-140 (GMP-140) also known as CD62P and P-selectin is present on the surface of platelets and endothelial cells, where it mediates platelet-leukocyte and endothelium-leukocyte interactions.
Recent work has established that these receptors share certain structural features. Each of the receptors in this class is a glycoprotein with a lectin-like domain, a region with homology to epidermal growth factor, and a region with homology to complement regulatory proteins (see, Springer (1989) Nature 346:425, which is incorporated herein by reference). The term "selectin" is used herein to refer to this class of lectin-like receptors.
There is currently an interest in developing highly specific competitive inhibitors of selectin-mediated cellular adhesion. Such inhibitors are useful in therapeutic regimens to treat various selectin-mediated disease responses. The inhibitors could also be used to target other pharmaceutical compounds, such as anti-inflammatory agents or anti-oxidants, to the sites of injury.
The broad participation of the selectins in inflammatory disease (Rosen & Bertozzi (1994) Curr. Op. Cell Biol. 6:663-673) has stimulated tremendous interest in the nature of their carbohydrate ligands as leads for the development of anti-inflammatory agents. All three selecting share a common recognition motif, the sialyl Lewis x (sLe.sup.x, NeuAc.alpha.2,3gal.beta.1,4(fuc.alpha.1,3)glcNAc) (FIG. 1) and the related sialyl Lewis a (sLe.sup.x, NeuAc.alpha.2,3gal.beta.1,3-(fuc.alpha.1,4)glcNAc) tetrasaccharides (Feizi (1993) Curr. Op. Struct. Biol. 3:701-710; Varki (1994) Proc. Natl. Acad. Sci. USA 91:7390-7397). Sialylation and fucosylation of the core N-acetyllactosamine disaccharide are essential for conferring binding activity. Although the interaction of the selecting with sLe.sup.x oligosaccharides is fairly weak, with equilibrium dissociation constants in the millimolar range (Cooke et al. (1994) Biochemistry 33:10591-10596; Jacob et al. (1995) Biochemistry 34:1210-1217), sLe.sup.x has proven to be an effective antagonist of selectin-mediated adhesion in several animal models of acute inflammation (Mulligan et al. (1993a) J. Exp. Med. 178:623-631; Mulligan et al. (1993b) Nature 364:149-151; Buerke et al. (1994) J. Clin. Invest. 93:1140-1148; Seekamp et al. (1994) Am. J. Pathol. 144:592-598).
Two central problems in the development of sLe.sup.x derivatives as anti-inflammatory agents are the lack of an inexpensive and expedient synthesis, and the susceptibility of the sialic acid and fuc.alpha.ose residues to enzymatic cleavage in the bloodstream (Uchiyama et al. (1995) J. Am. Chem. Soc. 117:5395-5396). The chemical syntheses of sLe.sup.x are laborious, involving difficult glycosylation reactions with sialic acid and fucose (Nicolaou et al. (1991) J. Chem. soc., Chem. Commun. 870-872; Nicolaou et al. (1992) J. Am. Chem. Soc. 114:3126-3128; Danishefsky et al. (1992a) J. Am. Chem. Soc. 114:8329-8331; Danishefsky et al. (1992b) J. Am. Chem. Soc. 114:8331-8334; Yoshida et al. (1993) Glycoconjugate J. 10:3-15; Stahl et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2096-2098). The enzymatic synthesis of sLe.sup.x is more efficient, but requires the use of enzymes and cofactors that are either not generally available or very expensive (Dumas et al. (1991) Bioorg. Med. Chem. Lett. 1:425-428; Ichikawa et al. (1992) J. Am. Chem. Soc. 114:9283-9898; Ball et al. (1992) J. Am. Chem. Soc. 114:5449; de Vries et al. (1993) FEBS Lett. 330:243-248; Palcic (1994) Meth. Enzymol. 230:300-316). These difficulties have prompted several groups to explore sLe.sup.x mimetics with reduced structural complexity, and with functional alternatives to sialic acid and fucose. For example, Chen et al. (1994) J. Biol. Chem. 269:1595-1598 and Brandley et al. (1993) Glycobiology 3:633-639, have demonstrated that replacement of sialic acid in sLe.sup.x with a sulfate ester results in equivalent or enhanced selectin binding activity. Uchiyama et al. (1995) supra, have incorporated the essential hydroxyl groups and anionic character of sLe.sup.x into simplified mimetics with equivalent E-selectin binding activity. Finally, a non-carbohydrate-based sLe.sup.x mimetic was designed based on the computer-identified structural similarity of a non-carbohydrate natural product (Rao et al. (1994) J. Biol. Chem. 269:19663-19666). See, also, U.S. Pat. No. 5,428,025, showing that N-acetyllactosamine ligands which bind to endothelial leukocyte adhesion molecule-1 (ELAM-1) are useful in relieving ELAM-1-mediated inflammation.